Legionella pneumophila (Lp) is the agent of Legionnaires'disease. It is ubiquitous in natural and man-made water systems, infecting individuals after aerosol inoculation. In aquatic habitats, Lp survives in biofilms and as an intracellular parasite of protozoa, whereas in the lung, it flourishes as a parasite of macrophages. Iron is vital to Lp growth in extra- and intracellular niches and its ability to cause disease. During this grant period, we discovered that Lp expresses a siderophore (legiobactin) that is novel in structure and regulation, the first such data in nearly 20 years of Lp research. We then demonstrated the ability of legiobactin-containing supernatants to stimulate the growth of iron-starved legionellae, purified the siderophore, and identified two genes (lbtAB) required for its expression. In other work, we characterized an Lp mutant that is highly defective for growth in low iron, macrophages, and the lung and showed that the inactivated gene (iraA) encodes a methyltransferase, whose predicted target is trans-aconitate, an inhibitor of aconitase. During this grant period, we also found that the cytochrome c maturation (ccm) operon plays a key role in Lp growth in low iron, in host cells, and in the mammalian lung. The role for iraA and ccrn in low iron and infection is without precedent. Finally, we identified an Lp ferrous transport system that is important for infection, a ferrioxidase that promotes iron assimilation, and an iron-dependent pigment that facilitates antibiotic formation. In this proposal, we will i) determine the role of legiobactin in vivo and in environmental situations, including biofilms, ii) seek the additional genes that encode the Lp siderophore, iii) determine if iraA indeed encodes a trans-aconitate methyltransferase that promotes infection by modulating aconitase, and iv) determine how the ccm locus promotes iron uptake and whether it does so by facilitating cytochrome maturation, heme export, or a novel undefined function. Taken together, these studies will increase our specific understanding of Lp physiology and pathogenesis as well as provide new general insight into siderophores and the mechanisms of iron acquisition, methyltransferases and aconitases as iron-responsive regulators, cytochromes and intracellular respiration, macrophage infection, virulence in the respiratory tract, and bacterial environmental survival in water, biofilms and low-temperature amoebae.